We investigated the role of Ca⁺⁺ and 3', 5'-cyclic AMP (cAMP) in epinephrine-induced lipolysis in rat fat cells using the fat cell perifusion system with several agents which have some influence on calcium transport. This perifusion system was of great advantage for observing minute-to-minute changes in glycerol and cAMP release from fat cells and the relationship between them.
Isolated fat cells were prepared from the epididymal adipose tissue of male Wistar strain rats by the method of Rodbell. Packed fat cells were placed in a plastic column (0.4 by 13.5cm) maintained at 37°C. Krebs-Ringer bicarbonate buffer (pH 7.4) containing 1% (W/V) bovine serum albumin, 2mM glucose and 1.27mM Ca⁺⁺ was used as the basal perifusion medium, and epinephrine, verapamil, lidocaine or 2, 4-dinitrophenol were added when necessary. The flow rate of the perifusion medium was 0.5 ml per min, and the perfusate collected every 2 min from the bottom of the column was assayed for glycerol and cAMP.
Fat cells were perifused for a 10-min equilibrium period, and over the next 10-min period several samples were taken for determination of basal rates of lipolysis. The infusion of 10^-5M epinephrine induced the plateau of glycerol release in 20 min by 41.8 n moles/2 min/100 mgTG, and its level was maintained. On the other hand, the level of cAMP release reached the maximal value of 110 f moles/2 min/100 mgTG in 10 min and decreased gradually. Omission of epinephrine from the perifusion medium made lipolysis return rapidly to the basal level.
The increase of Ca⁺⁺ concentration from 0 to 1.27 mM was enhanced by 38% in epinephrine-induced lipolysis; however, there was no change in cAMP release. The addition of verapamil, a potent Ca⁺⁺ antagonist, caused epinephrine-induced glycerol release to decrease, and moreover the 30-min verapamil pretreatment strongly inhibited epinephrine-induced lipolysis, but there was no effect on cAMP release. Lidocaine, a local anaesthetic, inhibited epinephrine-induced glycerol release, though it further elevated the cAMP release. 2, 4-Dinitrophenol, an oxidative phosphorylation inhibitor, completely blocked epinephrine-induced glycerol and cAMP release.
These data suggest that it isn't necessary to maintain a high level of intracellular cAMP in order to induce lipolysis, and that calcium ion plays an important role after cAMP production in lipolysis.

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Glucagon secretion was studied in rats with electrolytic lesions of the bilateral ventromedial hypothalamic area (VMH-L) under various experimental conditions.
The results obtained are as follows :
1. The basal plasma level of immunoreactive glucagon (IRG) was lowered in VMH-L rats 5 and 10 weeks after the operation. Plasma IRG levels after 24-hour starvation and during the arginine load were more significantly decreased in the VMH-L rats than in the control group.
2. The basal plasma level of immunoreactive insulin (IRI) showed significant positive correlations with body weight and Lee's index in these rats. The basal plasma level of IRG showed significant negative correlations with body weight, Lee's index and basal plasma IRI level.
3. In response to the arginine load, the plasma IRI level was significantly increased in VMH-L rats immediately after the operation, and the plasma IRG level was more significantly decreased in VMH-L rats 1 week after the operation than in the control group.
4. The response of plasma IRG to the arginine load was also lowered more in VMH-L rats than in rats pair-fed for 4 weeks after the operation.
5. 15 weeks after the operation, there was no significant difference in response of plasma IRI and IRG to the subcutaneous injection of epinephrine between VMH-L and control rats.
These findings indicate that hypoglucagonemia in the VMH-L rats was induced by various factors, such as disorder of the autonomic nervous system, excessive insulin release, etc. The impairment of glucagon secretion may contribute to the development of obesity observed in rats with VMH-lesions.

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We investigated the influence of age, sex, menstrual cycle and electrolyte balance on the renin-angiotensin-aldosterone (R-A-A) system in 94 healthy subjects, 40 males and 54 females between the ages of 16 and 101. Male and female pre- and postmenopausal groups were divided into 6 subgroups according to their age; G-1 : under 20 years of age, G-2 : 21 to 30, G-3 : 31 to 45, G-4 : 46 to 60, G-5 : 61 to 75 and G-6 : over 76. Premenopausal women were examined in both the follicular and luteal phases. The parameters analyzed were body height, body weight, body surface area (BSA), hematocrit, mean blood pressure (MBP), plasma renin activity (PRA), plasma aldosterone concentration (PAC), plasma electrolytes and urinary excretions of Na, K, creatinine and aldosterone (AER) measured in urine specimen collected over 2-3 hours in the morning. PRA, PAC and AER were measured by radioimmunoassay.
1) PRA and PAC decreased gradually with age in the subjects under 60 years of age. There were no further changes in PRA and PAC in men over 61 y.o. and in postmenopausal women, respectively.
2) In premenopausal women, PRA and PAC were significantly higher in the luteal phase than in the follicular phase.
3) Both PRA and PAC appeared slightly higher in men than in women during the follicular and postmenopausal phases, the differences not being significant. During the luteal phase, PRA and PAC were, however, significantly higher in women than in men.
4) Urinary excretion of Na (mEq/hr) was not significantly different among the groups except for G-1 vs. G-6 in men and the follicular phase vs. postmenopausal women in G-4. Urinary excretion of K and creatinine significantly decreased while plasma K concentraiton increased with age.
5) PRA correlated negatively with urinary excretion of Na or MBP and positively with urinary excretion of creatinine, hematocrit, body height, body weight and BSA. PAC correlated negatively with urinary excretion of Na or MBP and positively with urinary excretion of creatinine, hematocrit and body height.
These findings suggest that the decrease of PRA and PAC with age is not due to an increase in salt intake but rather reflects age-related changes in renal function. As to the evaluation of the R-A-A system, we should consider age, sex and stage of menstrual cycle in addition to salt intake, posture and circadian variations.

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Somatostatin analogs, which have been used as tracers in various somatostatin (GIF) radioimmunoassay systems such as N^α-tyrosyl-, [Tyr¹] - and [Tyr¹¹] -GIF, contain tryptophan at position eight in the structure. These peptides tend to be subject to oxidative degradation during the radioiodination procedure and labile under storage conditions. In the present study, we therefore developed a radioimmunoassay for GIF using ¹²⁵I- [Tyr⁸] - GIF as a labelled antigen which was a far more stable tracer.
[Tyr⁸] -GIF was radioiodinated by the lactoperoxidase method. The ¹²⁵I- [Tyr⁸] -GIF was purified on a CMC column chromatogram (1 × 8cm) by eluating either with the stepwise method of 0.002M (19ml) - 0.2M ammonium acetate, pH 4.6, or with the linear gradient method of 0.05M (50ml) -0.25M (50ml) ammonium acetate, pH 4.6. Each 2ml fraction was collected in polystyrene tubes. Immunoreactive ¹²⁵I- [Tyr⁸ J -GIF existed in the] _last peak under both elution conditions. The fractions containing ¹²⁵I- [Tyr⁸]-GIF in the ammonium acetate buffer were stored in the polystyrene tubes at -20°C until used. Under these conditions, ¹²⁵I_ [Tyr⁸] -GIF was completely adsorbed on the surface of the polystyrene tube, while free ¹²⁵I remained in the ammonium acetate buffer. Immediately before use, the stock solution was thawed, and the ammonium acetate buffer was discarded by eliminating free ¹²⁵I which had been liberated from the tracer during storage. Then, 1.0ml of buffer (0.1% gelatin-0.2% BSA-0.025M EDTA-0.15M NaC1-0.01M phophate, pH 7.4 : GBPBS) was added to the tube, which was vigorously shaken for 5 min with a ThermoMixer. Eighty to 90% of ¹²⁵I- [Tyr⁸] -GIF attached to the wall was recovered in GBPBS by this procedure. The repurification on the CMC column chromatogram of the recovered tracer in GBPBS after storage of one month revealed that 88.2% of applied radioactivity was eluated at the same position of ¹²⁵I- [Tyr⁸] -GIF.
An autoradiogram of enzymatic hydrolysates of ¹²⁵I- [Tyr⁸] -GIF showed that the labelled amino acid was 3-iodotyrosine. To elucidate the biological activity of iodinated [Tyr⁸]-GIF, [Tyr⁸]-GIF was iodinated with KI by the lactoperoxidase method. On the CMC column chromatogram, the products were divided into three fractions designated as Fr.I, Fr.II and Frill. Fr.II was identified as [3-iodotyrosine⁸] -GIF by comparison with the elution position of ¹²⁵I- [Tyr⁸] -GIF on the CMC column chromatogram. The in vivo potency of Fr.II to inhibit the release of TSH stimulated by TRH was 14.9% of GIF, indicating that the biological activity of iodinated [Tyr⁸] -GIF was markedly increased as compared with that of [Tyr⁸] -GIF (less than 0.5% of GIF).
Specific antiserum R 501 for GIF at the final dilution of 1 : 2,500 bound 39.3% of ¹²⁵I- [Tyr⁸] -GIF. The binding of the tracer to the antibody reached near the maximal level 19 hours after incubation at 4°C. For the separation of the free and bound tracers, the optimal incubation time with dextran coated charcoal was 15 minutes at 4°C. In the assay, glass tubes were better than polystyrene tubes because they lowered the non-specific binding of the free tracer.
Based on the above results, we performed the assay as follows : 0.2ml GBPBS, 0.1ml standard or sample solution, 0.1ml ¹²⁵I- [Tyr⁸] -GIF (7,000-10,000cpm) and 0.1ml antiserum R 501 (1 : 500) were combined in glass tubes. After the first incubation at 4°C for 48 hours, the free and bound tracers were separated by the dextran coated charcoal method or the double antibody method.

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Radioimmunoassay techniques have been widely used for the measurement of serum thyrotropin concentrations. However, in spite of the great sensitivity of these techniques, some normal thyrotropin concentrations are still less than the minimum detectable amount. Data set in which contents below a fixed value are undetectable is referred to as Type 1 censored sample, and the maximum likelihood method can be applicable to define the normal range in this type of data.
We have simultaneously measured serum thyrotropin concentrations obtained from 107 normal adult men by double antibody radioimmunoassay, and attempts were made to calculate the normal range by the maximum likelihood method.
Distribution fitting of the censored data was determined by conventional chi-square tests, and it was found that the data fitted a normal log distribution. Although the serum thyrotropin concentration was undetectable (less than 0.625μU/mL) in 14% of the subjects, a normal range could be calculated. Using one-side tolerance limits for 95% coverage of the population with 90% confidence, we obtained the normal range of thyrotropin as 0.39 to 4.8μU/mL with a mean value of 1.37μU/mL, and predicted that 74.8% of undetectable thyrotropin values will fall within the normal range calculated above.

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We established a radioreceptor assay for catecholamines (CA), utilizing the microsomal fraction of bovine myocardium as CA receptors and ³H-norepinephrine (³H-NA) as a ligand. Since ³H-NA binding to the prepared CA binding protein was inhibited by alpha-adrenergic blocking agents, the CA receptors used were assumed to be alpha-adrenergic receptors.
In this paper, we studied binding sites of CA to alpha-adrenergic receptors by a displacement study using such compounds as CA, CA metabolites, substances of dihydroxytetrahydronaphthalene derivatives, and other compounds.
Various compounds with catechol nucleus had an affinity to the CA binding protein.
The displacement study with compounds of dihydroxytetrahydronaphthalene derivatives revealed that compounds which were capable of binding to alpha-adrenoceptors had 2 neighbouring phenol groups in the benzen ring at either position 2 and 3, or at position 3 and 4. On the other hand, either phenylalanine or tyrosine, which has only one phenol group in the benzen ring, did not bind the receptors.
It is conceivable, therefore, that the binding sites of CA to alpha-adrenergic receptors is position 2 to 4 of the catechol nucleus, where 2 neighbouring phenol groups exist.

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Adrenal surgery was performed on 162 cases of various diseases, including 67 primary aldosteronism (PA), 22 adrenal Cushing's syndrome (ACS), 19 hypophyseal Cushing's syndrome (HCS) and 32 pheochromocytoma (Pheo), at Tohoku University School of Medicine from December 1956 to March 1976. The diagnosis, treatments and prognosis of these cases were surveyed.
The diagnostic accuracy of pneumoretroperitoneum with tomography was 63% in PA, 95% in ACS and 93% in Pheo, while that of adrenal scintigraphy was 86% in PA and 100% in ACS. Scintigraphy and aldosterone assay in venous blood samples were the most reliable for the localization of PA.
The unilateral posterior approach was the most suitable procedure for the removal of adrenal tumors in PA, ACS and single Pheo within a few hundred grams. The anterior approach was preferable for single Pheo over a few hundred grams and bilateral or multiple Pheo.
As regards the treatment for HCS, good results were obtained by a combination therapy of bilateral subtotal adrenalectomy, hypophyseal radiation and reserpine administration.
Two cases of Pheo were inoperable. There were 3 deaths immediately after the removal of Pheo. We later investigated the blood pressure in successfully treated cases. Hypertension was noticed in 18% of PA and 10% of ACS. No hypertensive case was found in Pheo.

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